spsA polynucleotides and polypeptides

ABSTRACT

spsA polypeptides and DNA (RNA) encoding such spsA and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such spsA for the treatment of infection, particularly bacterial infections. Antagonists against such spsA and their use as a therapeutic to treat infections, particularly bacterial infections are also disclosed. Also disclosed are diagnostic assays for detecting diseases related to the presence of spsA nucleic acid sequences and the polypeptides in a host. Also disclosed are diagnostic assays for detecting polynucleotides encoding spsA and for detecting the polypeptide in a host.

RELATED APPLICATIONS

[0001] This application claims benefit of U.S. patent applications Ser.Nos. 60/027,218, filed Sep. 30, 1996, 60/027,220, filed Oct. 1, 1996,and 60/027,075, filed Sep. 30, 1996.

FIELD OF THE INVENTION

[0002] This invention relates, in part, to newly identifiedpolynucleotides and polypeptides; variants and derivatives of thesepolynucleotides and polypeptides; processes for making thesepolynucleotides and these potypeptides, and their variants andderivatives and antagonists of the polypeptides; and uses of thesepolynucleotides, polypeptides, variants, derivatives and antagonists. Inparticular, in these and in other regards, the invention relates, topolynucleotides and polypeptides of spsA, hereinafter referred to as“spsA”.

BACKGROUND OF THE INVENTION

[0003] The majority of proteins that are translocated across one or moremembranes from the site of synthesis are initially synthesized with anN-terminal extension known as a signal, or leader, peptide (Wickner, W.,et al, (1991). Ann. Rev. Biochem. 60:101-124). Proteolytic cleavage ofthe signal sequence to yield the mature protein occurs during, orshortly after, the translocation event and is catalyzed in bothprokaryotes and eukaryotes by enzymes known as signal, or leader,peptidases (herein “SPases”). The bacterial SPases are membrane proteinsconsisting of a single polypeptide anchored to the membrane by one(Gram-positive (herein “G⁺”) and Gram-negative (herein G⁻) bacteria) ortwo (G⁻ bacteria) transmembrane sections. Predicted amino acid sequencesof bacterial SPases show a high level of similarity and are known forEscherichia coli (Wolfe, P. B., et at, (1983) J. Biol. Chem.258:12073-12080), Pseudomonas fluorescens (Black, M. T., et al, (1992).Biochem. J. 282:539-543), Salmonella typhimurium (van Dijl, J. M., etal, (1990). Mol. Gen. Genet. 223:233-240), Haemophilus influenzae(Fleischmann, R. D., et al, (1995). Science 269:496-512), Phormidiunlaminosum (Packer, J. C., et al, (1995). Plant Mol. Biol. 27:199-204. K.Cregg, et al: Signal peptidase from Staphylococcus aureus ManuscriptJB765-96), Bradyrhizobium japonicum (Müller, P , et al, (1995). Mol.Microbiol. 18:831-840), Rhodobacter capsulatus (Klug, G., et al, (1996).GenBank entry, accession number 268305), Bacillus subtilis (twochromosomal and two of plasmid origin (Akagawa, et al, (1995) Microbiol.141:3241-3245; Meljer. W. J J., et al, (1995). Mol Microbiol 17:621-631:van Dijl, J. M., et al, (1992). EMBO J. 11:2819-2828), Bacilluslicheniformis (Hoang, V., et al, (1993). Sequence P42668 submitted toembl/genbank/ddbj data banks.), Bacillus caldotyricus (van Dijl, J. M.(1993). Sequence p41027. submitted to embl/genbank/ddbj data banks),Bacillus amyloliquifaciens (two chromosomal genes) (Hoang, V. and J.Hofemeister. (1995). Biochim. Biophys. Acta 1269:64-68; van Dijl. J. M.(1993). Sequence p41026, submitted to embl/genbank/ddbj data banks) anda partial sequence has been reported for Bacillus pumilis (Hoang, V. andJ. Hofemeister. (1995). Biochim. Biophys. Acta 1269:64-68). Theseenzymes have been collectively defined as type-1 signal pepidases (vanDijl, J. M., et al, (1992). EMBO J. 11:2819-2828). Although the aminoacid sequences of fifteen bacterial SPases (and a sixteenth partialsequence) have now been reported, the best studied examples are leaderpeptidase (LPase or LepB) from E. coli and a SPase from B. subtilis(SipS).

[0004] It has been demonstrated that LPase activity is essential forcell growth in E. coli. Experiments whereby expression of the lepB gene,encoding LPase, was regulated either by a controllable ara promoter(Dalbey, P. E. and Wickner. 260:15925-15931) or by partial deletion ofthe natural promoter (Date, T. (1983). J. Bacteriol. 154:76-83)indicated that minimization of LPase production was associated withcessation of cell growth and division. In addition, an E. coli strainpossessing a mutated lepB gene (E. coli IT41) has been shown to have adrastically reduced growth rate and display a rapid and pronouncedaccumulation of preproteins when the temperature of the growth medium iselevated to 42° C. (Inada, T., et al, (1988). J. Bacteriol.171:585-587). These results imply that there is no other gene product inE. coli that can substitute for LPase and that lepB is a single-copygene in the E.coli chromosome. This is in contrast to at least twospecies within the G⁺ Bacillus genus, B. subtilis and B.amyloliquifaciens. It is known that there are at least two homologousSPase genes in each of these Bacillus species. The sipS gene can bedeleted from the chromosome of B. subtilis 168 without affecting cellgrowth rate or viability under laboratory conditions to yield a mutantstrain that can still process preα-amylase. A putative SPase sequence(Akagawa, et al, (1995) Microbiol. 141:3241-3245) may be thegene-product responsible for this activity and / or B. subtilis mayharbor more than two SPase genes. Two or more genes encoding distinctSPase homologues reside on the chromosome of the closely related speciesB. amyloiquifaciens (Hoang, V. and J. Hofemeister. (1995). Biochim.Biophys. Acta 1269:64-68) and there is evidence to suggest that B.Japonicum may possess more than one SPase (Müller, P. et al, (1995).Mol. Microbiol. 18:831-840; Müller, P., et al, (1995). Planta197:163-175). Although SPase sequences from seven genera of G+ bacteriaare now known, only the single Bacillus genus amongst the G+ eubacteriahas been investigated with respect to SPase characteristics. It wastherefore considered of interest to determine whether a G+ eubacteriumthat, unlike B. subtilis and B. amyloliquifaciens, is not known forexceptional secretion activity has genes encoding more than one SPasewith overlapping substrate specificities or whether it resembles E.coliand H. influenzae (and possibly other G- eubacteria)more closely in thatit has a single SPase gene. The recent publication of the entire genomicsequence of the obligate G⁺-like intracellular bacterium Mycoplasmagenitalium also reveals an interesting feature relating to heterogeneityamongst SPases (Fraser, C. M., et al, (1995). Science 270:397-403).Inhibitors of E. coli LPase have been reported (Allsop. A. E., et al,1995. Bioorg. & Med. Chem. Letts. 5:443-448).

[0005] Evidence has accumulated to suggest that LPase belongs to a newclass of serine protease that does not utilize a histamine as acatalytic base (Black, M. T., et al, (1992). Biochem. J. 282:539-543;Sung, M. and R. E. Dalbey. (1992). J. Biol. Chem 267:13154-13159) butmay instead employ a lysine side-chain to fulfill this role (Black, M.T. (1993). J. Bacteriol. 175:4957-4961; Tschantz. W. R., et al, (1993)J. Biol. Chem. 268:27349-27354). These observations and comparisons withLex A from E. coli led to the proposal that a serine-lysine catalyticdyad, similar to that thought to operate during peptide bond hydrolysiscatalyzed by LexA (Slilaty, S. N. and J. Little. (1987). Proc. Natl.Acad. Sci. USA 84:3987-3991), may operate in LPase (Black, M. T. (1993).J. Bacteriol. 175:4957-4961). Similar observations have since been madefor SPase from B. subtilis (van Dijl, J. M., et al, (1995). J. Biol.Chem. 270:3611-3618) and for the Tsp periplasmic protease from E. coli(Keiler, K. C. and R. T. Sauer. (1995). Biol. Chem. 270:28864-28868);the similarities of SipS to LexA have been suggested to extend toseveral regions of primary structure (van Dijl, J. M., et al, (1995). J.Biol. Chem. 270:3611-3618). The serine and lysine residues (90 and 145respectively in E. coli LPase numbering) known to be critical forcatalytic activity in both E. coli LPase (Black, M. T. (1993). J.Bacteriol. 175:4957-4961; Tschantz, W. R., et al, (1993) J. Biol. Chem.268:27349-27354) and B. subtilis SPase (van Dijl, J. M., et al, (1995).J. Biol. Chem. 270:3611-3618) and thought to form a catalytic dyad areboth conserved in the S. aureus protein SpsB (S36 and K77). In addition,the aspartate at position 155 (280 in E. coli LPase numbering) is alsoconserved (this residue appears important for activity of the SipS SPase(van Dijl, J. M., et al. (1995). J. Biol. Chem. 270:3611-3618) but lessso for LPase from E. coli (Sung, M. and R. E. Dalbey. (1992). J. Biol.Chem 267:13154-13159). The present invention provides a novel SPase fromS. aureus.

[0006] Clearly, there is a need for factors that may be used to screencompounds for antibiotic activity and which may also be used todetermine their roles in pathogenesis of infection, dysfunction anddisease. There is a need, therefore, for identification andcharacterization of such factors which can play a role in preventing,ameliorating or correcting infections, dysfunctions or diseases.

[0007] The polypeptide of the present invention has amino acid sequencehomology to known serine proteases.

SUMMARY OF THE INVENTION

[0008] Toward these ends, and others, it is an object of the presentinvention to provide polypeptides, inter alia, that have been identifiedas novel spsA by homology between the amino acid sequence set out inFIG. 2 and known amino acid sequences of other proteins such as Bacillussubtillis sipS.

[0009] It is a further object of the invention, moreover, to providepolynucleotides that encode spsA, particularly polynucleotides thatencode the polypeptide herein designated spsA.

[0010] In a particularly preferred embodiment of this aspect of theinvention the polynucleotide comprises the region encoding spsA in thesequence set out in FIG. 1 [SEQ ID NO:1], or a fragment, analogue orderivative thereof.

[0011] In another particularly preferred embodiment of the presentinvention there is a novel serine protease protein from Staphylococcusaureus comprising the amino acid sequence of FIG. 2 [SEQ ID NO:2], or afragment, analogue or derivative thereof.

[0012] In accordance with this aspect of the present invention there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible by the Staphylococcus aureus bacterial clone contained inNCIMB Deposit No. 40771.

[0013] In accordance with this aspect of the invention there areprovided isolated nucleic acid molecules encoding spsA, particularlyStaphylococcus spsA, including mRNAs, cDNAs, genomic DNAs and, infurther embodiments of this aspect of the invention includebiologically, diagnostically, prophylactically, clinically ortherapeutically useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives, and compositions comprising same.

[0014] In accordance with another aspect of the present invention, thereis provided the use of a polynucleotide of the invention for therapeuticor prophylactic purposes, in particular genetic immunization.

[0015] Among the particularly preferred embodiments of this aspect ofthe invention are naturally occurring allelic variants of spsA andpolypeptides encoded thereby.

[0016] In accordance with this aspect of the invention there areprovided novel polypeptides of Staphylococcus referred to herein as spsAas spell as biologically, diagnostically, prophylactically, clinicallyor therapeutically useful fragments, variants and derivatives thereof,variants and derivatives of the fragments, and analogs of the foregoing,and compositions comprising same.

[0017] Among the particularly preferred embodiments of this aspect ofthe invention are variants of spsA polypeptide encoded by naturallyoccurring alleles of the spsA gene.

[0018] In a preferred embodiment of this aspect of the invention thereare provided methods for producing the aforementioned spsA polypeptides.

[0019] In accordance with yet another aspect of the present invention,there are provided inhibitors to such polypeptides, useful asantibacterial agents, including, for example, antibodies.

[0020] In accordance with certain preferred embodiments of this aspectof the invention, there are provided products, compositions and methods.inter alia: assessing spsA expression, to treat upper respiratory tract(e.g. otitis media, bacterial tracheitis, acute epiglottitis,thyroiditis), lower respiratory (e.g. empyema, lung abscess), cardiac(e.g. infective endocarditis), gastrointestinal (e.g. secretorydiarrhoea, splenic abscess, retroperitoneal abscess), CNS (e.g. cerebralabscess), eye (e.g. blepharitis, conjunctivitis, keratitis,endophthalmitis, preseptal and orbital cellulitis, darcryocystitis),kidney and urinary tract (e.g. epididymitis, irtrarenal and perinephricabscess, toxic shock syndrome), skin (e.g. impetigo, folliculitis,cutaneous abscesses, cellulitis, wound infection, bacterial myositis)bone and joint (e.g. septic arthritis, osteomyelitis); assaying geneticvariation; and administering a spsA polypeptide or polynucleotide to anorganism to raise an immunological response against a bacteria,especially a Staphylococcus.

[0021] In accordance with certain preferred embodiments of this andother aspects of the invention there are provided polynucleotides thathybridize to spsA polynucleotide sequences.

[0022] In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against spsA polypeptides.

[0023] In accordance with yet another aspect of the present invention,there are provided spsA antagonists which are also preferablybacteriostatic or bacteriocidal.

[0024] In a further aspect of the invention there are providedcompositions comprising a spsA polynucleotide or a spsA polypeptide foradministration to a cell or to a multicellular organism.

[0025] Various changes and modifications within the spirit and scope ofthe disclosed invention will become readily apparent to those skilled inthe art from reading the following description and from reading theother parts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The following drawings depict certain embodiments of theinvention. They are illustrative only and do not limit the inventionotherwise disclosed herein

[0027]FIG. 1 shows the polynucleotide sequence of Staphylococcus aureusspsA [SEQ ID NO:1].

[0028]FIG. 2 shows the amino acid sequence of Staphylococcus aureus spsA[SEQ ID NO:2] deduced from the polynucleotide sequence of FIG. 1.

[0029]FIG. 3 shows the polynucleotide and deduced amino acid sequencesof Staphylococcus aureus spsA and spsB [SEQ ID NO:3].

GLOSSARY

[0030] The following illustrative explanations are provided tofacilitate understanding of certain terms used frequently herein,particularly in the Examples. The explanations are provided as aconvenience and are not limitative of the invention.

[0031] SPSA-BINDING MOLECULE, as used herein, refers to molecules orions which bind or interact specifically with spsA polypeptides orpolynucleotides of the present invention, including, for example enzymesubstrates, cell membrane components and classical receptors. Bindingbetween polypeptides of the invention and such molecules, includingbinding or binding or interaction molecules may be exclusive topolypeptides of the invention, which is preferred, or it may be highlyspecific for polypeptides of the invention, which is also preferred, orit may be highly specific to a group of proteins that includespolypeptides of the invention, which is preferred, or it may be specificto several groups of proteins at least one of which includes apolypeptide of the invention. Binding molecules also include antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

[0032] GENETIC ELEMENT generally means a polynucleotide comprising aregion that encodes a polypeptide or a polynucleotide region thatregulates replication, transcription or translation or other processesimportant to expression of the polypeptide in a host cell, or apolynucleotide comprising both a region that encodes a polypeptide and aregion operably linked thereto that regulates expression. Geneticelements may be comprised within a vector that replicates as an episomalelement; that is, as a molecule physically independent of the host cellgenome. They may be comprised within plasmids. Genetic elements also maybe comprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

[0033] HOST CELL is a cell which has been transformed or transfected, oris capable of transformation or transfection by an exogenouspolynucleotide sequence.

[0034] IDENTITY or SIMILARITY, as known in the art, are relationshipsbetween two or more polypeptide sequences or two or more polynucleotidesequences, as determined by comparing the sequences. In the art,identity also means the degree of sequence relatedness betweenpolypeptide or polynucleotide sequences, as the case may be, asdetermined by the match between strings of such sequences. Both identityand similarity can be readily calculated (Computational MolecularBiology, Lesk, A. M., ed , Oxford University Press, New York, 1988;Biocomputing Informatics and Genome Projects, Smith, D. W., ed.,Academic Press, New York, 1993: Computer Analysis of Sequence Data, PartI, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey,1994; Sequence Analysis in Molecular Biology, von Heinje, G., AcademicPress, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux,J., eds., M Stockton Press, New York, 1991). While there exist a numberof methods to measure identity and similarity between two polynucleotideor two polypeptide sequences, both terms are well known to skilledartisans (Sequence Analysis in Molecular Biology, von Heinje, G.,Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H.,and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonlyemployed to determine identity or similarity between sequences include,but are not limited to those disclosed in Carillo, H., and Lipman, D.,SIAM J. Applied Math., 48:1073 (1988). Preferred methods to determineidentity are designed to give the largest match between the sequencestested. Methods to determine identity and similarity are codified incomputer programs. Preferred computer program methods to determineidentity and similarity between two sequences include, but are notlimited to, GCG program package (Devereux, J., et al., Nucleic AcidsResearch 12(1). 387 (1984)), BLASTP, BLASTN, and FASTA Atschul, S. F. etal., J. Molec. Biol. 215: 403 (1990)).

[0035] ISOLATED means altered “by the hand of man” from its naturalstate; i.e., that, if it occurs in nature, it has been changed orremoved from its original environment, or both. For example, a naturallyoccurring polynucleotide or a polypeptide naturally present in a livingorganism in its natural state is not “isolated,” but the samepolynucleotide or polypeptide separated from the coexisting materials ofits natural state is “isolated”, as the term is employed herein. Forexample, with respect to polynucleotides, the term isolated means thatit is separated from the chromosome and cell in which it naturallyoccurs. As part of or following isolation, such polynucteotides can bejoined to other polynucleotides, such as DNAs, for mutagenesis, to formfusion proteins, and for propagation or expression in a host, forinstance. The isolated polynucleotides, alone or joined to otherpolynucleotides such as vectors, can be introduced into host cells, inculture or in whole organisms. Introduced into host cells in culture orin whole organisms, such DNAs still would be isolated, as the term isused herein, because they would not be in their naturally occurring formor environment. Similarly, the polynucleotides and polypeptides mayoccur in a composition, such as a media formulations solutions forintroduction of polynucleotides or polypeptides, for example, intocells, compositions or solutions far chemical or enzymatic reactions,for instance, which are not naturally occurring compositions, and,therein remain isolated polynucleotides or polypeptides within themeaning of that term as it is employed herein.

[0036] POLYNUCLEOTIDE(S) generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single-and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions or single-, double- andtriple-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded, or triple-stranded, or a mixture of single- anddouble-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including simple and complex cells, inter alia. Polynucleotidesembraces short polynucleotides often referred to as oligonucleotide(s).

[0037] POLYPEPTIDES, as used herein, includes all polypeptides asdescribed below. The basic structure of polypeptides is well known andhas been described in innumerable textbooks and other publications inthe art. In this context, the term is used herein to refer to anypeptide or protein comprising two or more amino acids joined to eachother in a linear chain by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types. It will be appreciated that polypeptides oftencontain amino acids other than the 20 amino acids commonly referred toas the 20 naturally occurring amino acids, and that many amino acids,including the terminal amino acids, may be modified in a givenpolypeptide, either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques which are well known to the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art.

[0038] Among the known modifications which may be present inpolypeptides of the present are, to name an illustrative few,acetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. Such modifications are well known to those of still andhave been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as, for instance PROTEINS—STRUCTURE AND MOLECULARPROPERTIES, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993). Many detailed reviews are available on this subject, suchas, for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pgs. 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York (1983); Seifter et al., Meth. Enzymol.182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, as is well known and as notedabove, that polypeptides are not always entirely linear. For instance,polypeptides may be generally as a result of posttranslation events,including natural processing event and events brought about by humanmanipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli or other cells, priorto proteolytic processing almost invariably will be N-formylmethionine.During post-translational modification of the peptide, a methionineresidue at the NH₂-terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing, and themethionineless amino terminal variants of the protein of the invention.The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as, forexample, E. coli . Accordingly, when glycosylation is desired, apolypeptide should be expressed in a glycosylating host, generally aeukaryotic cell. Insect cell often carry out the same posttranslationalglycosylations as mammalian cells and, for this reason, insect cellexpression systems have been developed to express efficiently mammalianproteins having native patterns of glycosylation, inter alia. Similarconsiderations apply to other modifications. It will be appreciated thatthe same type of modification may be present in the same or varyingdecree at several sites in a given polypeptide. Also, a givenpolypeptide may contain many types of modifications. In general, as usedherein, the term polypeptide encompasses all such modifications,particularly those that are present in polypeptides synthesizedrecombinantly by expressing a polynucleotide in a host cell.

[0039] VARIANT(S) of polynucleotides or polypeptides, as the term isused herein, are polynucleotides or polypeptides that differ from areference polynucleotide or polypeptide, respectively. Variants in thissense are described below and elsewhere in the present disclosure ingreater detail. (1) A polynucleotide that differs in nucleotide sequencefrom another, reference polynucleotide. Generally, differences arelimited so that the nucleotide sequences of the reference and thevariant are closely similar overall and, in many regions, identical. Asnoted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype a variant will encode a polypeptide with the same amino acidsequence as the reference. Also as noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below. (2) A polypeptide that differs in aminoacid sequence from another, reference polypeptide. Generally,differences are limited so that the sequences of the reference and thevariant are closely similar overall and, in many region, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention relates to novel spsA polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel spsA gene of Staphylococcus aureus, which isrelated by amino acid sequence homology to Bacillus subtillis sipSpolypeptide. The invention relates especially to spsA having thenucleotide and amino acid sequences set out in FIG. 1 and FIG. 2respectively, and to the spsA nucleotide and amino acid sequences of theDNA in NCIMB Deposit No. 40771, which is herein referred to as “thedeposited clone” or as the “DNA of the deposited clone.” It will beappreciated that the nucleotide and amino acid sequences set out inFIGS. 1 [SEQ ID NO:1] and 2 [SEQ ID NO:2] were obtained by sequencingthe DNA of the deposited clone. Hence, the sequence of the depositedclone is controlling as to any discrepancies between it (and thesequence it encodes) and the sequences of FIG. 1 [SEQ ID NO:1] and FIG.2 [SEQ ID NO:2].

[0041] Polynucleotides

[0042] In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the spsA polypeptidehaving the deduced amino acid sequence of FIG. 2 [SEQ ID NO:2].

[0043] Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1 [SEQ ID NO:1], a polynucleotide of thepresent invention encoding spsA polypeptide may be obtained usingstandard cloning and screening procedures, such as those for cloning andsequencing chromosomal DNA fragments from Staphylococcus aureus WCUH 29cells as starting material, followed by obtaining a full length clone.For example, to obtain a polynucleotide of the invention sequence, suchas that sequence given in FIG. 1 [SEQ ID NO:1] typically a library ofclones of chromosomal DNA of Staphylococcus aureus WCUH 29 in E. coli orsome other suitable host is probed with a radiolabelled oligonucleotide,preferably a 17-mer or longer, derived from a partial sequence. Clonescarrying DNA identical to that of the probe can then be distinguishedusing high stringency washes. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Conveniently such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.;Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).(see Screening By Hybridization 1.90 and Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Illustrative of the invention, thepolynucleotide set out in FIG. 1 [SEQ ID NO:1] was discovered in a DNAlibrary derived from Staphylococcus aureus WCUH 29.

[0044] SpsA of the invention is structurally related to other spsAproteins, as shown by the results of sequencing the DNA encoding spsA ofthe deposited clone. The DNA sequence thus obtained is set out in FIG. 1[SEQ ID NO:1]. It contains an open reading frame encoding a protein ofhaving about the number of amino acid residues set forth in FIG. 2 [SEQID NO:2] with a deduced molecular weight that can be calculated usingamino acid residue molecular weight values well known in the art. Theprotein exhibits greatest homology to Bacillus subtillis sipS proteinamong known proteins. spsA of FIG. 2 [SEQ ID NO:2] has homology with theamino acid sequence of Bacillus subtillis sipS.

[0045] Polynucleotides of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the anti-sense strand.

[0046] The coding sequence which encodes the polypeptide may beidentical to the coding sequence of the polynucleotide shown in FIG. 1[SEQ ID NO:1]. It also may be a polynucleotide with a differentsequence, which, as a result of the redundancy (degeneracy) of thegenetic code, encodes the polypeptide of FIG. 2 [SEQ ID NO:2].

[0047] Polynucleotides of the present invention which encode thepolypeptide of FIG. 2 [SEQ ID NO:2] may include, but are not limited tothe coding sequence for the mature polypeptide, by itself; the codingsequence for the mature polypeptide and additional coding sequences,such as those encoding a leader or secretory sequence, such a pre-, orpro- or prepro-protein sequence; the coding sequence of the maturepolypeptide with or without the aforementioned additional codingsequences together with additional, non-coding sequences, including forexample, but not limited to non-coding 5′ and 3′ sequences, such as thetranscribed, non-translated sequences that play a role in transcription(including termination signals, for example), ribosome binding, mRNAstability elements, and additional coding sequence which encodeadditional amino acids, such as those which provide additionalfunctionalities. Thus, for instance, the polypeptide may be fused to amarker sequence, such as a peptide, which facilitates purification ofthe fused polypeptide. In certain embodiments of this aspect of theinvention, the marker sequence is a hexa-histidine peptide, such as thetag provided in the pQE vector (Qiagen, Inc.), among others, many ofwhich are commercially available. As described in Gentz et al., Proc.Natl. Acad. Sci., USA 86: 821-824 (1989) for instance hexa-histidineprovides for convenient purification of the fusion protein. The HA tagmay also be used to create fusion proteins and corresponds to an epitopederived of influenza hemagglutinin protein, which has been described byWilson et al., i Cell 37: 767 (1984), for instance. Polynucleotides ofthe invention also include, but are not limited to, polynucleotidescomprising a structural gene and its naturally associated geneticelements.

[0048] In accordance with the foregoing, the term “polynucleotideencoding a polypeptide” as used herein encompasses polynucleotides whichinclude a sequence encoding a polypeptide of the present invention,particularly bacterial, and more particularly the Staphylococcus aureusspsA having the amino acid sequence set out in FIG. 2 [SEQ ID NO:2]. Theterm encompasses polynucleotides that include a single continuous regionor discontinuous regions encoding the polypeptide (for example,interrupted by integrated phage or insertion sequence or editing)together with additional regions, that also may contain coding and/ornon-coding sequences.

[0049] The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 2 [SEQ ID NO:2]. A variant of the polynucleotide may be a naturallyoccurring variant such as a naturally occurring allelic variant, or itmay be a variant that is not known to occur naturally. Suchnon-naturally occurring variants of the polynucleotide may be made bymutagenesis techniques, including those applied to polynucleotides,cells or organisms.

[0050] Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions

[0051] Among the particularly preferred embodiments of the invention inthis regard are polynucleotides encoding polypeptides having the aminoacid sequence of spsA set out in FIG. 2 [SEQ ID NO:2]; variants,analogs, derivatives and fragments thereof, and fragments of thevariants, analogs and derivatives.

[0052] Further particularly preferred in this regard are polynucleotidesencoding spsA variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of spsA polypeptide of FIG. 2 [SEQ ID NO:2] in whichseveral, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residuesare substituted, deleted or added, in any combination. Especiallypreferred among these are silent substitutions, additions and deletions,which do not alter the properties and activities of spsA. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polynucleotides encoding polypeptides having theamino acid sequence of FIG. 2 [SEQ ID NO:2], without substitutions.

[0053] Further preferred embodiments of the invention arepolynucleotides that are at least 70% identical over their entire lengthto a polynucleotide encoding spsA polypeptide having the amino acidsequence set out in FIG. 2 [SEQ ID NO:2], and polynucleotides which arecomplementary to such polynucleotides. Alternatively, most highlypreferred are polynucleotides that comprise a region that is at least80% identical over their entire length to a polynucleotide encoding spsApolypeptide of the Staphylococcus aureus DNA of the deposited clone andpolynucleotides complementary thereto. In this regard, polynucleotidesat least 90% identical over their entire length to the same areparticularly preferred, and among these particularly preferredpolynucleotides, those with at least 95% are especially preferred.Furthermore, those with at least 97% are highly preferred among thosewith at least 95%, and among these those with at least 98% and at least99% are particularly highly preferred, with at least 99% being the morepreferred.

[0054] Preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the DNA of FIG. 1 [SEQ ID NO:1].

[0055] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0056] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for RNA, cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingspsA and to isolate cDNA and genomic clones of other genes that have ahigh sequence similarity to the spsA gene. Such probes generally willcomprise at least 15 bases. Preferably, such probes will have at least30 bases and may have at least 50 bases. Particularly preferred probeswill have at least 30 bases and will have 50 bases or less.

[0057] For example, the coding region of the spsA gene may be isolatedby screening using the known DNA sequence to synthesize anoligonucleotide probe. A labeled oligonucleotide having a sequencecomplementary to that of a gene of the present invention is then used toscreen a library of cDNA, genomic DNA or mRNA to determine which membersof the library the probe hybridizes to.

[0058] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery oftreatments of and diagnostics for disease, particularly human disease,as further discussed herein relating to polynucleotide assays, interalia.

[0059] The polynucleotides of the invention that are oligonucleotides,including SEQ ID NOS:3 and 4, derived from the sequences of SEQ ID NOS:1and 2 may be used in the processes herein as described, but preferablyfor PCR, to determine whether or not the Staphylococcus aureus genesidentified herein in whole or in part are transcribed in infectedtissue. It is recognized that such sequences will also have utility indiagnosis of the stage of infection and type of infection the pathogenhas attained.

[0060] The polynucleotides may encode a polypeptide which is the matureprotein plus additional amino or carboxyl-terminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form, mayallow protein transport, may lengthen or shorten protein half-life ormay facilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

[0061] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0062] In sum, a polynucleotide of the present invention may encode amature protein, a mature protein plus a leader sequence (which may bereferred to as a preprotein), a precursor of a mature protein having oneor more prosequences which are not the leader sequences of a preprotein,or a preproprotein, which is a precursor to a proprotein, having aleader sequence and one or more prosequences, which generally areremoved during processing steps that produce active and mature forms ofthe polypeptide.

[0063] Deposited Materials

[0064] The deposit has been made under the terms of the Budapest Treatyon the International Recognition of the Deposit of Micro-organisms forPurposes of Patent Procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

[0065] A deposit containing a Staphylococcus aureus spsA bacterial clonehas been deposited with the National Collections of Industrial andMarine Bacteria Ltd. (NCIMB), 23 St. Machar Drive, Aberdeen AB2 1RY,Scotland on Sep. 11, 1995 and assigned NCIMB Deposit No. 40771. TheStaphylococcus aureus bacterial clone deposit is referred to herein as“the deposited clone” or as “the DNA of the deposited clone.”

[0066] The deposited material is a bacterial clone that contains thefull length spsA DNA, referred to as “NCIMB 40771” upon deposit.

[0067] The sequence of the polynucleotides contained in the depositedmaterial, as well as the amino acid sequence of the polypeptide encodedthereby are controlling in the event of any conflict with anydescription of sequences herein.

[0068] A license may be required to make, use or sell the depositedmaterials, and no such license is hereby granted.

[0069] Polypeptides

[0070] The present invention further relates to a spsA polypeptide whichhas a deduced amino acid sequence of 151 amino acids in length, as setforth in FIG. 2 [SEQ ID NO:2], and has a deduced molecular weight of21.692 kilodaltons.

[0071] The invention also relates to fragments, analogs and derivativesof these polypeptides. The terms “fragment,”“derivative” and “analog”when referring to the polypeptide of FIG. 2 [SEQ ID NO:2], means apolypeptide which retains-essentially the same biological function oractivity as such polypeptide. Thus, an analog includes a proproteinwhich can be activated by cleavage of the proprotein portion to producean active mature polypeptide.

[0072] A fragment, derivative or analog of the polypeptide of FIG. 2[SEQ ID NO:2] may be (i) one in which one or more of the amino acidresidues are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code, or(ii) one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be within the scope ofthose skilled in the art from the teachings herein.

[0073] Among the particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of spsA setout in FIG. 2 [SEQ ID NO:2], variants, analogs, derivatives andfragments thereof, and variants, analogs and derivatives of thefragments. Alternatively, particularly preferred embodiments of theinvention in this regard are polypeptides having the amino acid sequenceof the spsA, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments.

[0074] Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids AlaVal, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu substitution between theamide residues Asn and Gln, exchange of the basic residues Lvs and Argand replacements among the aromatic residues Phe, Tyr.

[0075] Further particularly preferred in this regard are variants,analogs. derivatives and fragments, and variants, analogs, andderivatives of the fragments having the amino acid sequence of the spsApolypeptide of FIG. 2 [SEQ ID NO:2], in which several, a few, 5 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the spsA. Also especially preferred in thisregard are conservative substitutions. Most highly preferred arepolypeptides having the amino acid sequence of FIG. 2 [SEQ ID NO:2]without substitutions.

[0076] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0077] The polypeptides of the present invention include the polypeptideof FIG. 2 [SEQ ID NO:2] (in particular the mature polypeptide) as wellas polypeptides which have at least 70% identity to the polypeptide ofFIG. 2 [SEQ ID NO:2], preferably at least 80% identity to thepolypeptide of FIG. 2 [SEQ ID NO:2], and more preferably at least 90%similarity (more preferably at least 90% identity) to the polypeptide ofFIG. 2 [SEQ ID NO:2] and still more preferably at least 95% similarity(still more preferably at least 95% identity) to the polypeptide of FIG.2 [SEQ ID NO:2] and also include portions of such polypeptides with suchportion of the polypeptide generally containing at least 30 amino acidsand more preferably at least 50 amino acids.

[0078] Fragments or portions of the polypeptides of the presentinvention may be employed for producing the corresponding full-lengthpolypeptide by peptide synthesis; therefore, the fragments may beemployed as intermediates for producing the full-length polypeptides.Fragments or portions of the polynucleotides of the present inventionmay be used to synthesize full-length polynucleotides of the presentinvention.

[0079] Fragments

[0080] Also among preferred embodiments of this aspect of the presentinvention are polypeptides comprising fragments of spsA, mostparticularly fragments of spsA having the amino acid set out in FIG. 2[SEQ ID NO2], and fragments of variants and derivatives of the spsA ofFIG. 2 [SEQ ID NO:2].

[0081] In this regard a fragment is a polypeptide having an amino acidsequence that entirely is the same as part but not all of the amino acidsequence of the aforementioned spsA polypeptides and variants orderivatives thereof.

[0082] Such fragments may be “free-standing,” i.e., not part of or fusedto other amino acids or polypeptides, or they may be comprised within alarger polypeptide of which they form a part or region. When comprisedwithin a larger polypeptide, the presently discussed fragments mostpreferably form a single continuous region. However, several fragmentsmay be comprised within a single larger polypeptide. For instance,certain preferred embodiments relate to a fragment of a spsA polypeptideof the present comprised within a precursor polypeptide designed forexpression in a host and having heterologous pre and pro-polypeptideregions fused to the amino terminus of the spsA fragment and anadditional region fused to the carboxyl terminus of the fragments.Therefore, fragments in one aspect of the meaning intended herein,refers to the portion or portions of a fusion polypeptide or fusionprotein derived from spsA.

[0083] Representative examples of polypeptide fragments of theinvention, include, for example, fragments from amino acid number 1-20,21-40, 41-60, 61-80, 81-100, and 101-151, and any combination of these20 amino acid fragments.

[0084] In this context “about” herein includes the particularly recitedranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidat either extreme or at both extremes.

[0085] Preferred fragments of the invention include, for example,truncation polypeptides of spsA. Truncation polypeptides include spsApolypeptides having the amino acid sequence of FIG. 2, or of variants orderivatives thereof, except for deletion of a continuous series ofresidues (that is, a continuous region, part or portion, at includes theamino terminus, or a continuous series of residues that includes thecarboxyl terminus or, as in double truncation mutants, deletion of twocontinuous series of residues, one including the amino terminus and oneincluding the carboxyl terminus. Fragments having the size ranges setout about also are preferred embodiments of truncation fragments, whichare especially preferred among fragments generally. Degradation forms ofthe polypeptides of the invention in a host cell, particularly aStaphylococcis, are also preferred.

[0086] Also preferred in this aspect of the invention are fragmentscharacterized by structural or functional attributes of spsA. Preferredembodiments of the invention in this regard include fragments thatcomprise alpha-helix and alpha-helix forming regions, beta-sheet andbeta-sheet-forming regions, turn and turn-forming regions, coil andcoil-forming regions, hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions, substrate binding region, and high antigenicindex regions of spsA, and combinations of such fragments.

[0087] Preferred regions are those that mediate activities of spsA. Mosthighly preferred in this regard are fragments that have a chemical,biological or other activity of spsA, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Further preferred polypeptide fragments are those that areantigenic or immunogenic in an animal, especially in a human.

[0088] It will be appreciated that the invention also relates to amongothers, polynucleotides encoding the aforementioned fragments,polynucleotides that hybridize to polynucleotides encoding thefragments, particularly those that hybridize under stringent conditions,and polynucleotides, such as PCR primers, for amplifying polynucleotidesthat encode the fragments. In these regards, preferred polynucleotidesare those that correspond to the preferred fragments, as discussedabove.

[0089] Vectors, Host Cells, Expression

[0090] The present invention also relates to vectors which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polypeptides of the invention by recombinant techniques.

[0091] Host cells can be genetically engineered to incorporatepolynucleotides and express polypeptides of the present invention.Introduction of a polynucleotides into the host cell can be affected bycalcium phosphate transfection, DEAE-dextran mediated transfection,transvection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULARBIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING: A LABORATORYMANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

[0092] Polynucleotide constructs in host cells can be used in aconventional manner to produce the gene product encoded by therecombinant sequence. Alternatively, the polypeptides of the inventioncan be synthetically produced by conventional peptide synthesizers.

[0093] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989).

[0094] In accordance with this aspect of the invention the vector maybe, for example, a plasmid vector, a single or double-stranded phagevector, a single or double-stranded RNA or DNA viral vector. Plasmidsgenerally are designated herein by a lower case p preceded and/orfollowed by capital letters and/or numbers, in accordance with standardnaming conventions that are familiar to those of skill in the art.Starting plasmids disclosed herein are either commercially available,publicly available, or can be constructed from available plasmids byroutine application of well known published procedures. Many plasmidsand other cloning and expression vectors that can be used in accordancewith the present invention are well known and readily available to thoseof skill in the art.

[0095] Preferred among vectors, in certain respects, are those forexpression of polynucleotides and polypeptides of the present invention.Generally, such vectors comprise cis-acting control regions effectivefor expression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting, factors either are supplied by thehost supplied by a complementing vector or supplied by the vector itselfupon introduction into the host.

[0096] In certain preferred embodiments in this regard, the vectorsprovide for specific expression. Such specific expression may beinducible expression or expression only in certain types of cells orboth inducible and cell-specific. Particularly preferred among induciblevectors are vectors that can be induced for expression by environmentalfactors that are easy to manipulate, such as temperature and nutrientadditives. A variety of vectors suitable to this aspect of theinvention, including constitutive and inducible expression vectors foruse in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

[0097] A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include, among others,chromosomal episomal and virus-derived vectors. e.g., vectors derivedfrom bacterial plasmids from bacteriophage from transposons, from yeastepisomes, from insertion elements, from yeast chromosomal elements, fromviruses such as baculoviruses, papova viruses, such as SV40, vacciniaviruses, adenoviruses, fowl pox viruses, pseudorabies viruses andretroviruses, and vectors derived from combinations thereof, such asthose derived from plasmid and bacteriophage genetic elements, such ascosmids and phagemids, all may be used for expression in accordance withthis aspect of the present invention. Generally, any vector suitable tomaintain, propagate or express polynucleotides to express a polypeptidein a host may be used for expression in this regard.

[0098] The appropriate DNA sequence may be inserted into the vector byany of a variety of well-known and routine techniques, such as, forexample, those set forth in Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0099] The DNA sequence in the expression vector is operatively linkedto appropriate expression control sequence(s), including, for instance,a promoter to direct mRNA transcription. Representatives of suchpromoters include, but are limited to, the phage lambda PL promoter, theE. coli lac, trp and tac promoters, the SV40 early and late promotersand promoters of retroviral LTRs.

[0100] In general, expression constructs will contain sites fortranscription initiation and termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0101] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, un accordance withmany commonly practiced procedures, such regions will operate bycontrolling transcription, such as transcription factors, repressorbinding sites and termination, among others.

[0102] Vectors for propagation and expression generally will includeselectable markers and amplification regions, such as, for example,those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y. (1989).

[0103] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, streptomyces andBacillus subtilis cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 andBowes melanoma cells; and plant cells.

[0104] The following vectors, which are commercially available, areprovided by way of example. Among vectors preferred for use in bacteriaare pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,pRIT5 available from Pharmacia, and pBR322 (ATCC 37017). Among preferredeukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG availablefrom Stratagene; and pSVK3, pBPV, pMSG and pSVL available fromPharmacia. These vectors are listed solely by way of illustration of themany commercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

[0105] Promoter regions can be selected from any desired gene usingvectors that contain a reporter transcription unit lacking a promoterregion, such as a chloramphenicol acetyl transferase (“CAT”)transcription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the catgene engenders production of CAT activity, which can be detected bystandard CAT assays. Vectors suitable to this end are well known andreadily available, such as pKK232-8 and pCM7. Promoters for expressionof polynucleotides of the present invention include not only well knownand readily available promoters, but also promoters that readily may beobtained by the foregoing technique, using a reporter gene.

[0106] Among known prokaryotic promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli lacI and lacZ and promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

[0107] Among known eukaryotic promoters suitable in this regard are theCMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), and metallothionein promoters,such as the mouse metallothionein-I promoter.

[0108] Recombinant expression vectors will include, for example, originsof replication, a promoter preferably derived from a highly-expressedgene to direct transcription of a downstream structural sequence, and aselectable marker to permit isolation of vector containing cells afterexposure to the vector.

[0109] Polynucleotides of the invention, encoding the heterologousstructural sequence of a polypeptide of the invention generally will beinserted into the vector using standard techniques so that it isoperably linked co the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG or GTG that initiates translation of thepolypeptide to be expressed. Generally, there will be no other openreading frames that begin with an initiation codon, usually AUG, and liebetween the ribosome binding site and the initiation codon. Also,generally, there will be a translation stop codon at the end of thepolypeptide and there will be a polyadenylation signal in constructs foruse in eukaryotic hosts. Transcription termination signal appropriatelydisposed at the 3′ end of the transcribed region may also be included inthe polynucleotide construct.

[0110] For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polypeptide. These signals may beendogenous to the polypeptide or they may be heterologous signals.

[0111] The polypeptide may be expressed in a modified form, such as afusion protein, and may include not only secretion signals but alsoadditional heterologous functional regions. Thus, for instance, a regionof additional amino acids, particularly charged amino acids, may beadded to the N-terminus of the polypeptide to improve stability andpersistence in the host cell, during purification or during subsequenthandling and storage. Also, region also may be added to the polypeptideto facilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologrous region from immunoglobulin that is useful to solubilize orpurify polypeptides. For example, EP-A-O 464 533 (Canadian counterpart2045869) discloses fusion proteins comprising various portions ofconstant region of immunoglobin molecules together with another proteinor part thereof. In drug discovery, for example, proteins have beenfused with antibody Fc portions for the purpose of high-throughputscreening assays to identify antagonists. See, D. Bennett et al..Journal of Molecular Recognition, Vol 8 52-58 (1995) and K. Johanson etal., The Journal of Biological Chemistry, Vol. 270, No. 16, pp 9459-9471(1995).

[0112] Cells typically then are harvested by centrifugation, disruptedby physical or chemical means, and the resulting crude extract retainedfor further purification.

[0113] Microbial cells employed in expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing, agents, suchmethods are well know to those skilled in the art.

[0114] Mammalian expression vectors may comprise an origin ofreplication, a suitable promoter and enhancer, and also any necessaryribosome binding sites, polyadenylation regions, splice donor andacceptor sites, transcriptional termination sequences, and 5′ flankingnon-transcribed sequences that are necessary for expression.

[0115] spsA polypeptide can be recovered and purified from recombinantcell cultures by well-known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polypeptide is denatured during isolation and or purification.

[0116] Polynucleotide Assays

[0117] This invention is also related to the use of the spsApolynucleotides to detect complementary polynucleotides such as, forexample, as a diagnostic reagent. Detection of spsA in a eukaryote,particularly a mammal and especially a human, will provide a diagnosticmethod for diagnosis of a disease. Eukaryotes (herein also“individual(s)”), particularly mammals, and especially humans,particularly those infected with an organism comprising the spsA genemay be detected at the DNA level by a variety of techniques. Nucleicacids for diagnosis may be obtained from an infected individual's cellsand tissues, such as bone, blood, muscle, cartilage, and skin. GenomicDNA may be used directly for detection or may be amplified enzymaticallyby using PCR (Saiki et al., Nature, 324: 163-166 (1986) prior toanalysis. RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding spsA can be usedto identify and analyze spsA presence and for expression. Using PCR,characterization of the strain of prokaryote present in a eukaryote,particularly a mammal, and especially a human, may be made by ananalysis of the genotype of the prokaryote gene. For example, deletionsand insertions can be detected by a chance in size of the amplifiedproduct in comparison to the genotype of a reference sequence. Pointmutations can be identified by hybridizing amplified DNA toradiolabelled spsA RNA or alternatively, radiolabelled spsA antisenseDNA sequences. Perfectly matched sequences can be distinguished frommismatched duplexes by RNase A digestion or by differences in meltingtemperatures.

[0118] Sequence differences between a reference gene and genes havingmutations also may be revealed by direct DNA sequencing. In addition,cloned DNA segments may be employed as probes to detect specific DNAsegments. The sensitivity of such methods can be greatly enhanced byappropriate use of PCR or another amplification method. For example, asequencing primer is used with double-stranded PCR product or asingle-stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabelled nucleotide or by automatic sequencing procedures withfluorescent-tags.

[0119] Genetic characterization based on DNA sequence differences may beachieved by detection of alteration in electrophoretic mobility of DNAfragments in gels, with or without denaturing agents. Small sequencedeletions and insertions can be visualized by high resolution gelelectrophoresis. DNA fragments of different sequences may bedistinguished on denaturing formamide gradient gels in which themobilities of different DNA fragments are retarded in the gel atdifferent positions according to their specific melting or partialmelting temperatures (see, e.g., Myers et al., Science, 230: 1242(1985)).

[0120] Sequence changes at specific locations also may be revealed bynuclease protection assays, such as RNase and S 1 protection or thechemical cleavage method (e.g., Cotton et al., Proc. Natl. Acad. Sci.,USA, 85: 4397-4401 (1985)).

[0121] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, e.g.,restriction fragment length polymorphisms (RFLP) and Southern blottingof genomic DNA.

[0122] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations also can be detected by in situ analysis.

[0123] Cells carrying mutations or polymorphisms in the gene of thepresent invention may also be detected at the DNA level by a variety oftechniques, to allow for serotyping, for example. For example, RT-PCRcan be used to detect mutations. It is particularly preferred to usedRT-PCR in conjunction with automated detection systems, such as, forexample, GeneScan. RNA or cDNA may also be used for the same purpose,PCR or RT-PCR. As an example, PCR primers complementary to the nucleicacid encoding spsA can be used to identify and analyze mutations. Theseprimers may be used for amplifying spsA DNA isolated from a samplederived from an individual.

[0124] The invention provides a process for diagnosing, disease,preferably bacterial infections, more preferably Staphylococcus aureusand most preferably upper respiratory tract (e.g. otitis media,bacterial tracheitis, acute epiglottitis, thyroiditis), lowerrespiratory (e.g. empyema, lung abscess), cardiac (e.g. infectiveendocarditis), Gastrointestinal (e.g. secretory diarrhoea, splenicabscess, retroperitoneal abscess), CNS (e.g. cerebral abscess), eye(e.g. blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptaland orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) bone and joint (e.g. septicarthritis, osteomyelitis), comprising determining from a sample derivedfrom an individual a increased level of expression of polynucleotidehaving the sequence of FIG. 1 [SEQ ID NO:1]. Increased expression ofspsA polynucleotide can be measured using any one of the methods wellknown in the art for the quantation of polynucleotides, such as, forexample, PCR, RT-PCR, RNase protection, Northern blotting and otherhybridization methods.

[0125] Polypeptide Assays

[0126] The present invention also relates to a diagnostic assays such asquantitative and diagnostic assays for detecting levels of spsA proteinin cells and tissues, including determination of normal and abnormallevels. Thus, for instance, a diagnostic assay in accordance with theinvention for detecting over-expression of spsA protein compared tonormal control tissue samples may be used to detect the presence of aninfection, for example. Assay techniques that can be used to determinelevels of a spsA protein, in a sample derived from a host are well-knownto those of skill in the art. Such assay methods includeradioimmunoassays, competitive-binding assays, Western Blot analysis andELISA assays. Among these ELISAs frequently are preferred. An ELISAassay initially comprises preparing an antibody specific to spsA,preferably a monoclonal antibody. In addition a reporter antibodygenerally is prepared which binds to the monoclonal antibody. Thereporter antibody is attached a detectable reagent such as radioactive,fluorescent or enzymatic reagent, in this example horseradish peroxidaseenzyme.

[0127] Antibodies

[0128] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as an immunogen toproduce antibodies thereto. The present invention includes, for examplesmonoclonal and polyclonal antibodies, chimeric, single chain andhumanized antibodies, as well as Fab fragments, or the product of an Fabexpression library.

[0129] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptides can be used to generate antibodiesbinding the whole native polypeptides. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0130] For preparation of monoclonal antibodies, any technique known inthe art which provides antibodies produced by continuous cell linecultures can be used. Examples include various techniques, such as thosein Kohler, G. and Milstein, C., Nature 256: 495-497 (1975); Kozbor etat., Immunology Today 4: 72 (1983); Cole et al., pg. 77-96 in MONOCLONALANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc. (1985).

[0131] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice, or other organisms such as other mammals, may beused to express humanized antibodies to immunogenic polypeptide productsof this invention.

[0132] Alternatively phage display technology could be utilised toselect antibody genes with binding activities towards the polypeptideeither from repertoires of PCR amplified v-genes of lymphocytes fromhumans screened for possessing anti-Fbp or from naive libraries(McCafferty, J. et al., (1990), Nature 348, 552-554; Marks, J. et al.,(1992) Biotechnology 10, 779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352, 624-628).

[0133] If two antigen binding domains are present each domain may bedirected against a different epitope—termed ‘bispecific’ antibodies.

[0134] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or purify the polypeptide ofthe present invention by attachment of the antibody to a solid supportfor isolation and/or purification by affinity chromatography.

[0135] Thus, among others, antibodies against spsA may be employed toinhibit and/or treat infections, particularly bacterial infections andespecially upper respiratory tract (e.g. otitis media, bacterialtracheitis, acute epiglottitis, thyroiditis), lower respiratory (e.g.empyema, lung abscess), cardiac (e.g. infective endocarditis),gastrointestinal (e.g. secretory diarrhoea, splenic abscess,retroperitoneal abscess), CNS (e.g. cerebral abscess), eye (e.g.blepharitis, conjunctivitis, keratitis, endophthalmitis, preseptal &orbital cellulitis, darcryocystitis), kidney and urinary tract (e.g.epididymitis, intrarenal and perinephric abscess, toxic shock syndrome),skin (e.g. impetigo, folliculitis, cutaneous abscesses, cellulitis,wound infection, bacterial myositis) and bone and joint (e.g septicarthritis, osteomyelitis).

[0136] Polypeptide derivatives include antigenically, epitopically orimmunologically equivalent derivatives which form a particular aspect ofthis invention. The term “antigenically equivalent derivative” as usedherein encompasses a polypeptide or its equivalent which will bespecifically recognised by certain antibodies which, when raised to theprotein or polypeptide according to the present invention, interferewith the immediate physical interaction between pathogen and mammalianhost. The term “immunologically equivalent derivative” as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between pathogen andmammalian host.

[0137] The polypeptide, such as an antigenically or immunologicallyequivalent derivative or a fusion protein thereof is used as an antigento immunize a mouse or other animal such as a rat or chicken. The fusionprotein may provide stability to the polypeptide. The antigen may beassociated, for example by conjugation, with an immunogenic carrierprotein for example bovine serum albumin (BSA) or keyhole limpethaemocyanin (KLH). Alternatively a multiple antigenic peptide comprisingmultiple copies of the protein or polypeptide, or an antigenically orimmunologically equivalent polypeptide thereof may be sufficientlyantigenic to improve immunogenicity so as to obviate the use of acarrier.

[0138] Preferably the antibody or derivative thereof is modified to makeit less immunogenic in the individual. For example, if the individual ishuman the antibody may most preferably be “humanised”; where thecomplimentarity determining region(s) of the hybridoma-derived antibodyhas been transplanted into a human monoclonal antibody, for example asdescribed in Jones, P. et al. (1986), Nature 321, 522-525 or Tempest etal.,(1991) Biotechnology 9, 266-273.

[0139] The use of a polynucleotide of the invention in geneticimmunization will preferably employ a suitable delivery method such asdirect injection of plasmid DNA into muscles (Wolff et al., Hum MolGenet 1992, 1:363, Manthorpe et al., Hum. Gene Ther. 1963:4, 419),delivery of DNA complexed with specific protein carriers ( Wu et al., JBiol Chem 1989:264,16985), coprecipitation of DNA with calcium phosphate(Benvenisty & Reshef, PNAS,1986:83,9551), encapsulation of DNA invarious forms of liposomes (Kaneda et al., Science 1989:243,375),particle bombardment (Tang et al., Nature 1992, 356:152, Eisenbraun etal., DNA Cell Biol 1993, 12:791) and in vivo infection using clonedretroviral vectors (Seeger et al., PNAS 1984:81,5849).

[0140] spsA-binding Molecules and Assays

[0141] This invention also provides a method for identification ofmolecules, such as binding molecules, that bind spsA. Genes encodingproteins that bind spsA. can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Such methods are described in many laboratory manuals such as, forinstance. Coligan et al, Current Protocols in Immunology 1(2): Chapter 5(1991). Also, a labeled ligand can be photoaffinity linked to a cellextract.

[0142] Polypeptides of the invention also can be used to assess spsAbinding capacity of spsA-binding molecules, in cells or in cell-freepreparations.

[0143] Polypeptides of the invention may also be used to assess thebinding or small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural (productmixtures. These substrates and ligands may be natural substrates andligands of may be structural functional mimetics.

[0144] Antagonists and-Assays and Molecules

[0145] The invention also provides a method of screening compounds toidentify those which block (antagonist) the action of spsA polypeptidesor polynucleotides, such as its interaction with spsA-binding molecules.

[0146] For example, to screen for antagonists, a synthetic reaction mix,a cellular compartment, such as a membrane, cell envelope or cell wall,or a preparation of any thereof, may be prepared from a cell thatexpresses a molecule that binds sps. The preparation is incubated withlabeled spsA in the absence or the presence of a candidate moleculewhich may be a spsA antagonist. The ability of the candidate molecule tobind the binding molecule is reflected in decreased binding of thelabeled ligand. Molecules which bind gratuitously, i.e., withoutinducing the effects of spsA on binding the spsA binding molecule, aremost likely to be good antagonists.

[0147] spsA-like effects of potential antagonists may by measured, forinstance, by determining activity of a reporter system followinginteraction of the candidate molecule with a cell or appropriate cellpreparation, and comparing the effect with that of spsA or moleculesthat elicit the same effects as spsA. Reporter systems that may beuseful in this regard include but are not limited to colorimetriclabeled substrate converted into product, a reporter gene that isresponsive to changes in spsA activity, and binding assays known in theart.

[0148] Another example of an assay for spsA antagonists is a competitiveassay that combines spsA and a potential antagonist with membrane-boundspsA-binding molecules, recombinant spsA binding molecules, naturalsubstrates or ligands, or substrate or ligand mimetics, underappropriate conditions for a competitive inhibition assay. spsA can belabeled, such as by radioactivity or a colorimetric compound, such thatthe number of spsA molecules bound to a binding molecule or converted toproduct can be determined accurately to assess the effectiveness of thepotential antagonist.

[0149] Potential antagonists include small organic molecules, peptides.polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptiled a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducingspsA-induced activities, thereby preventing the action of spsA byexcluding spsA from binding.

[0150] Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such that normal biological activity isprevented. Examples of small molecules include but are not limited tosmall organic molecules, peptides or peptide-like molecules.

[0151] Other potential antagonists include antisense molecules (seeOkano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES AS ANTISENSEINHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton. Fla. (1988), for adescription of these molecules).

[0152] Preferred potential antagonists include compounds related to andderivatives of spsA.

[0153] In a particular aspect the invention provides the use of thepolypeptide, polynucleotide or inhibitor of the invention to interferewith the initial physical interaction between a pathogen and mammalianhost responsible for sequelae of infection. In particular the moleculesof the invention may be used: i) in the prevention of adhesion ofbacteria, in particular gram positive bacteria, to mammalianextracellular matrix proteins on in-dwelling devices or to extracellularmatrix proteins in wounds; ii) to block serine protease protein mediatedmammalian cell invasion by, for example, initiating phosphorylation ofmammalian tyrosine kinases (Rosenshine et al., Infect. Immun. 60:2211(1992); iii) to block bacterial adhesion between mammalian extracellularmatrix proteins and bacterial serine protease proteins which mediatetissue damage; iv) to block the normal progression of pathogenesis ininfections initiated other than by the implantation of in-dwellingdevices or by other surgical techniques.

[0154] Each of the DNA sequences provided herein may be used in thediscovery and development of antibacterial compounds. The encodedprotein upon expression can be used as a target for the screening ofantibacterial drugs. Additionally, the DNA sequences encoding the aminoterminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct antisense sequences to control the expression of the codingsequence of interest.

[0155] The antagonists may be employed for instance to inhibit upperrespiratory tract (e.g. otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis, lower respiratory (e.g. empyema, lungabscess), cardiac (e.g. infective endocarditis), gastrointestinal (e.g.secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS(e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal and orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g. epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g. septic arthritis,osteomyelitis).

[0156] Vaccines

[0157] Another aspect of the invention relates to a method for inducingan immunological response in an individual, particularly a mammal whichcomprises inoculating the individual with spsA, or a fragment or variantthereof, adequate to produce antibody to protect said individual frominfection, particularly bacterial infection and most particularlyStaphylococcus infections. Yet another aspect of the invention relatesto a method of inducing immunological response in an individual whichcomprises, through gene therapy, delivering gene encoding spsA, or afragment or a variant thereof, for expressing spsA, or a fragment or avariant thereof in vivo in order to induce an immunological response toproduce antibody to protect said individual from disease.

[0158] A further aspect of the invention relates to an immunologicalcomposition which, when introduced into a host capable of having inducedwithin it an immunological response, induces an immunological responsein such host to a spsA or protein coded therefrom, wherein thecomposition comprises a recombinant spsA or protein coded therefromcomprising DNA which codes for and expresses an antigen of said spsA orprotein coded therefrom.

[0159] The spsA or a fragment thereof may be fused with co-protein whichmay not by itself produce antibodies, but is capable of stabilizing thefirst protein and producing a fused protein which will have immunogenicand protective properties. Thus fused recombinant protein, preferablyfurther comprises an antigenic co-protein, such asGlutathione-S-transferase (GST) or beta-galactosidase, relatively largeco-proteins which solubilise the protein and facilitate production andpurification thereof. Moreover, the co-protein may act as an adjuvant inthe sense of providing a generalized stimulation of the immune system.The co-protein may be attached to either the amino or carboxy terminusof the first protein.

[0160] Provided by this invention are methods using the describedpolynucleotide or particular fragments thereof which have been shown toencode non-variable regions of bacterial cell surface proteins in DNAconstructs used in such genetic immunization experiments in animalmodels of infection with Staphylococcus aureus will be particularlyuseful for identifying protein epitopes able to provoke a prophylacticor therapeutic immune response. It is believed that this approach willallow for the subsequent preparation of monoclonal antibodies ofparticular value from the requisite organ of the animal successfullyresisting or clearing infection for the development of prophylacticagents or therapeutic treatments of Staphylococcus aureus infection inmammals, particularly humans.

[0161] The polypeptide may be used as an antigen for vaccination of ahost to produce specific antibodies which protect against invasion ofbacteria, for example by blocking adherence of bacteria to damagedtissue. Examples of tissue damage include wounds in skin or connectivetissue caused e.g. by mechanical, chemical or thermal damage or byimplantation of indwelling devices, or wounds in the mucous membranes,such as the mouth, mammary glands, urethra or vagina.

[0162] The present invention also includes a vaccine formulation whichcomprises the immunogenic recombinant protein together with a suitablecarrier. Since the protein may be broken down in the stomach, it ispreferably administered parenterally, including, for example,administration that is subcutaneous, intramuscular, intravenous, orintradermal. Formulations suitable for parenteral administration includeaqueous and non-aqueous sterile injection solutions which may containanti-oxidants, buffers, bacteriostats and solutes which render theformulation instonic with the bodily fluid, preferably the blood, of theindividual: and aqueous and non-aqueous sterile suspensions which mayinclude suspending agents or thickening agents. The formulations may bepresented in unit-dose or multi-dose containers, for example, sealedampoules and vials and may be stored in a freeze-dried conditionrequiring only the addition of the sterile liquid carrier immediatelyprior to use. The vaccine formulation may also include adjuvant systemsfor enhancing the immunogenicity of the formulation, such as oil-inwater systems and other systems known in the art. The dosage will dependon the specific activity of the vaccine and can be readily determined byroutine experimentation.

[0163] While the invention has been described with reference to certainspsA embodiments, it is to be understood that this covers fragments ofthe naturally occurring protein and similar proteins with additions,deletions or substitutions which do not substantially affect theimmunogenic properties of the recombinant protein.

[0164] Compositions

[0165] The invention also relates to compositions comprising thepolynucleotide or the polypeptides discussed above or the antagonists.Thus, the polypeptides of the present invention may be employed incombination with a non-sterile or sterile carrier or carriers for usewith cells, tissues or organisms, such as a pharmaceutical carriersuitable for administration to a subject. Such compositions comprise,for instance, a media additive or a therapeutically effective amount ofa polypeptide of the invention and a pharmaceutically acceptable carrieror excipient. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, water, glycerol, ethanol and combinationsthereof. The formulation should suit the mode of administration.

[0166] Kits

[0167] The invention further relates to diagnostic and pharmaceuticalpacks and kits comprising one or more containers filled with one or moreof the ingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

[0168] Administration

[0169] Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

[0170] The pharmaceutical compositions may be administered in anyeffective, convenient manner including, for instance, administration bytopical, oral, anal, vaginal, intravenous, intraperitoneal,intramuscular, subcutaneous, intranasal or intradermal routes amongothers.

[0171] The pharmaceutical compositions generally are administered in anamount effective for treatment or prophylaxis of a specific indicationor indications. In general, the compositions are administered in anamount of at least about 10 μg/kg body weight. In most cases they willbe administered in an amount not in excess of about 8 mg/kg body weightper day. Preferably, in most cases, dose is from about 10 μg/kg to about1 mg/kg body weight, daily. It will be appreciated that optimum dosagewill be determined by standard methods for each treatment modality andindication, taking into account the indication, its severity, route ofadministration, complicating conditions and the like.

[0172] In therapy or as a prophylactic, the active agent may beadministered to an individual as an injectable composition, for exampleas a sterile aqueous dispersion, preferably isotonic.

[0173] Alternatively the composition may be formulated for topicalapplication for example in the form of ointments, creams, lotions, eyeointments, eye drops, ear drops, mouthwash, impregnated dressings andsutures and aerosols, and may contain appropriate conventionaladditives, including, for example, preservatives, solvents to assistdrug penetration, and emollients in ointments and creams. Such topicalformulations may also contain compatible conventional carriers, forexample cream or ointment bases, and ethanol or oleyl alcohol forlotions. Such carriers may constitute from about 1% to about 98% byweight of the formulation; more usually they will constitute up to about80% by weight of the formulation.

[0174] For administration to mammals, and particularly humans. it isexpected that the daily dosage level of the active agent will be from0.01 mg/kg to 10 mg/kg, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual and will vary with the age, weight and response of theparticular individual. The above dosages are exemplary of the averagecase. There can, of course, be individual instances where higher orlower dosage ranges are merited, and such are within the scope of thisinvention.

[0175] In-dwelling devices include surgical implants, prosthetic devicesand catheters, i.e., devices that are introduced to the body of anindividual and remain in position for an extended time. Such devicesinclude, for example, artificial joints, heart valves, pacemakers,vascular grafts, vascular catheters, cerebrospinal fluid shunts, urinarycatheters, continuous ambulatory peritoneal dialysis (CAPD) catheters,etc.

[0176] The composition of the invention may be administered by injectionto achieve a systemic effect against relevant bacteria shortly beforeinsertion of an in-dwelling device. Treatment may be continued aftersurgery during the in-body time of the device. In addition, thecomposition could also be used to broaden perioperative cover for anysurgical technique to prevent Staphylococcus wound infections.

[0177] Many orthopaedic surgeons consider that humans with prostheticjoints should be considered for antibiotic prophylaxis before dentaltreatment that could produce a bacteraemia. Late deep infection is aserious complication sometimes leading to loss of the prosthetic jointand is accompanied by significant morbidity and mortality. It maytherefore be possible to extend the use of the active agent as areplacement for prophylactic antibiotics in this situation.

[0178] In addition to the therapy described above, the compositions ofthis invention may be used generally as a wound treatment agent toprevent adhesion of bacteria to matrix proteins exposed in wound tissueand for prophylactic use in dental treatment as an alternative to, or inconjunction with, antibiotic prophylaxis.

[0179] Alternatively, the composition of the invention may be used tobathe an indwelling device immediately before insertion. The activeagent will preferably be present at a concentration of 1 μg/ml to 10mg/ml for bathing of wounds or indwelling devices.

[0180] A vaccine composition is conveniently in injectable form.Conventional adjuvants may be employed to enhance the immune response.

[0181] A suitable unit dose for vaccination is 0.5-5 μg/kg of antigen,and such dose is preferably administered 1-3 times and with an intervalof 1-3 weeks.

[0182] With the indicated dose range, no adverse toxicological effectswill be observed with the compounds of the invention which wouldpreclude their administration to suitable individuals.

[0183] The antibodies described above may also be used as diagnosticreagents to detect the presence of bacteria containing the serineprotease protein.

[0184] All references and patent applications disclosed herein areincorporated by reference herein in their entirety.

EXAMPLES

[0185] The present invention is further described by the followingexamples. These exemplification's, while illustrating certain specificaspects of the invention, do not portray the limitations or circumscribethe scope of the disclosed invention.

[0186] Certain terms used herein are explained in the foregoingglossary.

[0187] The following bacterial strains were used in these Examples: E.coli XL-1-Blue, JM109 and lT41, S. aureus RN4220, H (ATCC13801), Oxford(ATCC9144) and WCUH29 (NCIMB 40771). S. aureus strains were known intryptone soya broth (TSB, Oxoid) or in Luria-Bertani broth (Sambrook,J., et al, (1989). Molecular Cloning: a Laboratory manual. 2^(nd) edCold Spring, Harbor Laboratory Press Cold Spring Harbor N.Y.). E. coliwas cultured in Luria-Bertani broth. For solid medium. 1 5% w/v) agarwas added. Where appropriate, the medium was supplemented with 100 μg mlampicillin for E. coli and 5 μg ml chloramphenicol for S. aureus. The S.aureus WCUH29 genomic library was constructed by Stratagene in thevector λZapII. The λZapII s. aureus H library was constructed in thislaboratory according to the instructions of the manufacturers of λZapII(Stratagene). All shuttle vector constructs that were transferred fromE. coli to S. aureus WCUH29 were first used to transform therestriction-minus S. aureus strain RN4220 and plasmid purified therefrom was used to transform S. aureus WCUH29. Plasmids used in this studyare described in the examples below.

Example 1

[0188] DNA Cloning

[0189] (A) DNA techniques and materials

[0190] Plasmid DNA was isolated using the RPM Kit (Bio 101 Inc.) or theWizard Midiprep DNA Purification System (Promega). PCR products wereisolated by horizontal agarose gel electrophoresis, treated with Agarase(Boehringer Mannheim) and purified with the Wizard DNA Clean-Up System(Promega). Chromosomal DNA was isolated from E. coli and S. aureus usingeither the Genomic DNA Purification Kit (Bacterial) (Advanced GeneticTechnologies Corp.) or following published procedures (Marmur, J.(1961). J. Mol. Biol. 3:208-218). An incubation and the appropriatequantity of lysotaphin (Applied Microbiology Inc.) was included duringthe preparation of plasmid and chromosomal DNA from S. aureus tofacilitate cell lysis. λZapII library clones were excised andrecircularized to form recombinant phagermid as described in themanufacturer's instructions. Procedures for DNA restriction anddephophorylation, agarose gel electrophoresis, PCR and transformation ofcompetent E. coli cells were performed essentially as described inSambrook et al. (Sambrook, J., et al. (1989). Molecular Cloning: aLaboratory manual. 2^(nd) ed Cold Spring Harbor Laboratory Press ColdSprings Harbor N.Y.). Electrocompetent S. aureus cells were prepared asdescribed by Schenk and Laddaga (Schenk, S. and R. A. Laddaga. 1992.Micorobiol. Letts. 94:133-138) with the following modifications: thebacteria were grown in TSB and washed at 2500×g for 5 times at 20° C.Competent cells and plasmid DNA were electroporated in 1 mm gapelectroporation cuvettes at 20° C., 100 Ω, 25 μF, and 2.3 kV in a GenePulser apparatus with pulse controller (Bio-Rad Laboratories Ltd.).Restriction enzymes and calf intestine alkaline phosphatase werepurchased from Promega, ligations were performed using a DNA LigationKit (Amersham) as described in the manufacturer's instructions,sequencing was performed using the Sequence Version 2.0 DNA SequencingKit (Amersham). PCR reactions, optimized using the Opti-Prime Kit(Stratagene), were performed on the Hybaid OmniGene thermal cycler usingeither Pfu DNA polymerase (Promega) and the products cloned using theTa-Cloning Kit (Invitrogen), the pGEM-T Cloning Kit (Promega) or the DNALigation Kit (Amersham). Oligonucleotides were synthesized by CruachemLtd or R&D Systems Europe Ltd. PCR primer sequences [SEQ ID NO:4-13],with intentional differences from template sequence shown in upper case,were as follows: Primer A(g/a)tIgg(a/t)(c/t)tIcc(a/t)gg(a/t)ga(a/t)aI(a/t)(g/a)t [SEQ ID NO:4](Primer A RtIggWYtIccWggWgaWaIWRt [SEQ ID NO:4]) Primer Bc(t/g)(a/g)tt(a/g)tc(t/a)cccatIa(t/c)(a/g)aa(a/g)ta [SEQ ID NO:5](Primer B cKRttRtcWcccatIaYRaaRta [SEQ ID NO:5]) Primer CtgGAaTTCAtgaaaaaagaaCtGttggaatggattatttc [SEQ ID NO:6] Primer DatttgtAAGCtTttaGtttttGgtGttttcaggattgaaa [SEQ ID NO:7] Primer EctgGATCCcgcttgattagttttattga [SEQ ID NO:8] Primer FttGGtACCttttgacacctctttttaag [SEQ ID NO:9] Primer GaaGGtACCtatgaaacaaatacaacatc [SEQ ID NO:10] Primer HatGAAtTCtcaatataattgtgacactc [SEQ ID NO:11] Primer I atattagagcgataattcc[SEQ ID NO:12] Primer J gttcatttgctattcttc [SEQ ID NO:13]

[0191] (B) PCR Cycle Conditions for Primer Pairs

[0192] A+B[SEQ ID NO:4 AND 5]5 min at 94° C, 30 cycles of [1 min at 94°C. 1 min at 42° C., 1 min at 72° C. ], 5 mins at 72° C.

[0193] C+D [SEQ ID NO:6 AND 7]: 5 mins at 94° C. 15 cycles of [1 min at94° C., 1 min at 50° C., 1 min at 72° C.], 5 mins at 72° C.

[0194] E+F [SEQ ID NO:8 AND 9]: 5 mins at 94° C., 15 cycles of [1 min at94° C., 1 min at 60° C., 2 mins at 72° C.], 5 mins at 72° C.

[0195] G+H [SEQ ID NO:10 AND 11]: 5 mins at 94° C. 15 cycles of [1 minat 94° C., 1 min at 45° C., 2 mins at 72° C., 2 mins at 72° C.], 5 minsat 72° C.

[0196] I+J [SEQ ID NO:12 AND 13]: 5 mins at 94° C., 30 cycles of [1 minat 94° C., 1 min at 42° C., 3 mins at 72° C.], 5 mins at 72° C.

[0197] DNA was Southern blotted from 0.7% (w/v) agarose gels onto nylonmembranes (Hybond-N′, Amersham) as described in the manufacturer'sinstructions. For library screening purposes plaques were transferred tonylon membranes (Hybond-N, Amersham) as described in the manufacturer'sinstructions. Membranes were hybridized with either oligonucleotide orwhole gene probes labeled using the ECL 3′-oligolabelling kit or the ECLrandom prime labeling kit (Amersham) as appropriate. Washing anddetection steps were performed as described in the manufacturer'sinstructions. All sequence data manipulation was performed with theWisconsin Package of Genetics Computer Group.

[0198] (C) PCR Cloning

[0199] A strategy was devised to clone SPase by PCR using primers A+B[SEQ ID NO:4 AND 5] as described above. A fragment of DNA of theexpected length, based upon the known size of SPases from Bacillus, wasobtained from a preparation of genomic DNA from S. aureus Oxford. The163 bp of sequence so derived is highly homologous to 163 bp of the sipSgene from B. subtillis implying that a fragment of a gene encoding atype-I SPase had been cloned. The PCR product was labeled and used toprobe a λZap-II library of S. aureus WCUH29 genomic DNA in order toobtain full sequence information. A positive clone was identified andthe recombinant plasmid (pKC10) excised, cut with several restrictionenzymes, blotted and probed with the same labeled DNA fragment asaforementioned. A SaII-digested pKC10 preparation was religated to formplasmid pKC11. The DNA sequence of 3093 bp of insert DNA was determinedby oligonucleotide walking in both directions from within the sequenceoriginally derived by PCR. FIG. 3 [SEQ ID NO:3] shows 1220 nucleotidesof DNA sequence from the chromosome of S. aureus WCUH29 of whichnucleotides 817-1025 represent the probe region. The probe sequencecomprises part of a potential open reading frame (ORF) which in itsentirely encodes a polypeptide of 151 amino acid residues with acalculated molecular mass of 21, 692 Da and with a single section ofhydrophobic residues close to the N-terminus that probably form a singletransmembrane anchor. The small surface-exposed domain and singletransmembrane anchor are typical of SPases from G+ eubacteria. Thepredicted protein has high homology with all known G+SPases and the genehas been named spsB (signal peptidase from Staphylococcus). It isnoteworthy that the highest levels oF sequence similarity are in regionsof the protein corresponding to the most highly conserved regions ofknown SPases. All three of these regions within the B. subtilis SPase,and two within LPase from E. coli contain at least one residue that iscritical for catalytic activity (Black, M. T. (1993). J. Bacteriol.175:4957-4961; Tschantz, W. R., et al, (1993) J. Biol. Chem.268:27349-27354; van Dijl, J. M., et al, (1995). J. Biol. Chem.270:3611-3618). A second ORF, named spsA, proximal to the spsB gene(separated by 15 nucleotides) putatively encodes a protein of 174 aminoacid residues with a calculated molecular mass of 20,146 Da. Sequencecomparison reveals that this protein in also similar to known SPasesequences. An optimized alignment of SpsB with SpsA results in 62%similarly and 31% identify between the two sequences. The regions ofhighest sequence conservation are concentrated within or close together.However, a surprising observation is the fact that neither the serinenor the lysine residue known to be essential for catalytic activity inknown type-I SPases are conserved in SpsA. In order to ascertain thatthe existence of spsA is not peculiar to the WCUH29 strain of S. aureusa λZapII library of DNA isolated from S. aureus strain H was probed withthe probe originally used to identify clones containing the spsAlB genesfrom strain WCUH29. Close homologues of both spsA and spsB were alsodiscovered in S. aureus H; SpsA homologues also lack active-site serineand lysine residues.

1 13 525 base pairs nucleic acid single linear Genomic DNA 1 GTGAAAAAAGTTGTAAAATA TTTGATTTCA TTGATACTTG CTATTATCAT TGTACTGTTC 60 GTACAAACTTTTGTAATAGT TGGTCATGTC ATTCCGAATA ATGATATGTC GCCAACCCTT 120 AACAAAGGGGATCGTGTTAT TGTAAATAAA ATTAAAGTTA CATTTAATCA ATTGAATAAT 180 GGTGATATCATTACATATAG GCGTGGTAAC GAGATATATA CTAGTCGAAT TATTGCCAAA 240 CCTGGTCAATCAATGGCGTT TCGTCAGGGA CAATTATACC GTGATGACCG ACCGGTTGAC 300 GCATCTTATGCCAAGAACAG AAAAATTAAA GATTTTAGTT TGCGCAATTT TAAAGAATTA 360 GATGGAGATATTATACCGCC TAACAATTTT GTTGTGCTAA ATGATCATGA TAACAATCAG 420 CATGATTCTAGACAATTTGG TTTAATTGAT AAAAAGGATA TTATTGGTAA TATAAGTTTG 480 AGATATTATCCTTTTTCAAA ATGGACGATT CAGTTCAAAT CTTAA 525 174 amino acids amino acidsingle linear protein 2 Met Lys Lys Val Val Lys Tyr Leu Ile Ser Leu IleLeu Ala Ile Ile 1 5 10 15 Ile Val Leu Phe Val Gln Thr Phe Val Ile ValGly His Val Ile Pro 20 25 30 Asn Asn Asp Met Ser Pro Thr Leu Asn Lys GlyAsp Arg Val Ile Val 35 40 45 Asn Lys Ile Lys Val Thr Phe Asn Gln Leu AsnAsn Gly Asp Ile Ile 50 55 60 Thr Tyr Arg Arg Gly Asn Glu Ile Tyr Thr SerArg Ile Ile Ala Lys 65 70 75 80 Pro Gly Gln Ser Met Ala Phe Arg Gln GlyGln Leu Tyr Arg Asp Asp 85 90 95 Arg Pro Val Asp Ala Ser Tyr Ala Lys AsnArg Lys Ile Lys Asp Phe 100 105 110 Ser Leu Arg Asn Phe Lys Glu Leu AspGly Asp Ile Ile Pro Pro Asn 115 120 125 Asn Phe Val Val Leu Asn Asp HisAsp Asn Asn Gln His Asp Ser Arg 130 135 140 Gln Phe Gly Leu Ile Asp LysLys Asp Ile Ile Gly Asn Ile Ser Leu 145 150 155 160 Arg Tyr Tyr Pro PheSer Lys Trp Thr Ile Gln Phe Lys Ser 165 170 1220 base pairs nucleic acidsingle linear Genomic DNA 3 TAGAACAGCA TTTTATGGGA TCGAAAAAGG AGTGACATCGTGAAAAAAGT TGTAAAATAT 60 TTGATTTCAT TGATACTTGC TATTATCATT GTACTGTTCGTACAAACTTT TGTAATAGTT 120 GGTCATGTCA TTCCGAATAA TGATATGTCG CCAACCCTTAACAAAGGGGA TCGTGTTATT 180 GTAAATAAAA TTAAAGTTAC ATTTAATCAA TTGAATAATGGTGATATCAT TACATATAGG 240 CGTGGTAACG AGATATATAC TAGTCGAATT ATTGCCAAACCTGGTCAATC AATGGCGTTT 300 CGTCAGGGAC AATTATACCG TGATGACCGA CCGGTTGACGCATCTTATGC CAAGAACAGA 360 AAAATTAAAG ATTTTAGTTT GCGCAATTTT AAAGAATTAGATGGAGATAT TATACCGCCT 420 AACAATTTTG TTGTGCTAAA TGATCATGAT AACAATCAGCATGATTCTAG ACAATTTGGT 480 TTAATTGATA AAAAGGATAT TATTGGTAAT ATAAGTTTGAGATATTATCC TTTTTCAAAA 540 TGGACGATTC AGTTCAAATC TTAAAAAGAG GTGTCAAAATTGAAAAAAGA ATTATTGGAA 600 TGGATTATTT CAATTGCAGT CGCTTTTGTC ATTTTATTTATAGTAGGTAA ATTTATTGTT 660 ACACCATATA CAATTAAAGG TGAATCAATG GATCCAACTTTGAAAGATGG CGAGCGAGTA 720 GCTGTAAACA TTATTGGATA TAAAACAGGT GGTTTGGAAAAAGGTAATGT AGTTGTCTTC 780 CATGCAAACA AAAATGATGA CTATGTTAAA CGTGTCATCGGTGTTCCTGG TGATAAAGTA 840 GAATATAAAA ATGATACATT ATATGTCAAT GGTAAAAAACAAGATGAACC ATATTTAAAC 900 TATAATTTAA AACATAAACA AGGTGATTAC ATTACTGGGACTTTCCAAGT TAAAGATTTA 960 CCGAATGCGA ATCCTAAATC AAATGTCATT CCAAAAGGTAAATATTTAGT TCTTGGAGAT 1020 AATCGTGAAG TAAGTAAAGA TAGCCGTGCG TTTGGCCTCATTGATGAAGA CCAAATTGTT 1080 GGTAAAGTTT CATTTAGATT CTGGCCATTT AGTGAATTTAAACATAATTT CAATCCTGAA 1140 AATACTAAAA ATTAATATGA AACAAATACA ACATCGTTTGTCGGTTTTAA TACTGATAAA 1200 CGATGTTTTA TTTTGTTAGT 1220 23 base pairsnucleic acid single linear Genomic DNA 4 RTNGGWYTNC CWGGWGAWAN WRT 23 23base pairs nucleic acid single linear Genomic DNA 5 CKRTTRTCWCCCATNAYRAA RTA 23 40 base pairs nucleic acid single linear Genomic DNA 6TGGAATTCAT GAAAAAAGAA CTGTTGGAAT GGATTATTTC 40 40 base pairs nucleicacid single linear Genomic DNA 7 ATTTGTAAGC TTTTAGTTTT TGGTGTTTTCAGGATTGAAA 40 28 base pairs nucleic acid single linear Genomic DNA 8CTGGATCCCG CTTGATTAGT TTTATTGA 28 28 base pairs nucleic acid singlelinear Genomic DNA 9 TTGGTACCTT TTGACACCTC TTTTTAAG 28 28 base pairsnucleic acid single linear Genomic DNA 10 AAGGTACCTA TGAAACAAAT ACAACATC28 28 base pairs nucleic acid single linear Genomic DNA 11 ATGAATTCTCAATATAATTG TGACACTC 28 19 base pairs nucleic acid single linear GenomicDNA 12 ATATTAGAGC GATAATTCC 19 18 base pairs nucleic acid single linearGenomic DNA 13 GTTCATTTGC TATTCTTC 18

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of: (a) a polynucleotide having atleast a 70% identity to a polynucleotide encoding a polypeptidecomprising amino acids 1 to 174 of SEQ ID NO:2; (b) a polynucleotidewhich is complementary to the polynucleotide of (a); and (c) apolynucleotide comprising at least 15 sequential bases of thepolynucleotide of (a) or (b).
 2. The polynucleotide of claim 1 whereinthe polynucleotide is DNA.
 3. The polynucleotide of claim 1 wherein thepolynucleotide is RNA.
 4. The polynucleotide of claim 2 comprisingnucleotides set forth in SEQ ID NO:1 encoding spsA.
 5. Thepolynucleotide of claim 2 comprising spsA coding sequence set forth inSEQ ID NO:1.
 6. The polynucleotide of claim 2 which encodes apolypeptide comprising amino acid 1 to 174 of SEQ ID NO:2.
 7. Anisolated polynucleotide comprising a member selected from the groupconsisting of: (a) a polynucleotide having at least a 70% identity to apolynucleotide encoding the same mature polypeptide expressed by the DNAcontained in NCIMB Deposit No. 40771 and having the polynucleotidesequence of SEQ ID NO:1; (b) a polynucleotide complementary to thepolynucleotide of (a); and (c) a polynucleotide comprising at least 15bases of the polynucleotide of (a) or (b).
 8. A vector comprising theDNA of claim
 2. 9. A host cell comprising the vector of claim
 8. 10. Aprocess for producing a polypeptide comprising: expressing from the hostcell of claim 9 a polypeptide encoded by said DNA.
 11. A process forproducing a cell which expresses a polypeptide comprising transformingor transfecting the cell with the vector of claim 8 such that the cellexpresses the polypeptide encoded by the cDNA contained in the vector.12. A polypeptide comprising an amino acid sequence which is at least70% identical to amino acid 1 to 174 of SEQ ID NO:2.
 13. A polypeptidecomprising an amino acid sequence as set forth in SEQ ID NO:2.
 14. Anantibody against the polypeptide of claim
 12. 15. An antagonist whichinhibits the activity of the polypeptide of claim
 12. 16. A method forthe treatment of an individual having need of spsA comprising:administering to the individual a therapeutically effective amount ofthe polypeptide of claim
 12. 17. The method of claim 16 wherein saidtherapeutically effective amount of the polypeptide is administered byproviding to the individual DNA encoding said polypeptide and expressingsaid polypeptide in vivo.
 18. A method for the treatment of anindividual having need to inhibit spsA polypeptide comprising:administering to the individual a therapeutically effective amount ofthe antagonist of claim
 15. 19. A process for diagnosing a diseaserelated to expression of the polypeptide of claim 12 comprising:determining a nucleic acid sequence encoding said polypeptide.
 20. Adiagnostic process comprising: analyzing for the presence of thepolypeptide of claim 12 in a sample derived from a host.
 21. A methodfor identifying compounds which bind to and inhibit an activity of thepolypeptide of claim 12 comprising: contacting a cell expressing on thesurface thereof a binding for the polypeptide, said binding beingassociated with a second component capable of providing a detectablesignal in response to the binding of a compound to said binding, with acompound to be screened under conditions to permit binding to thebinding; and determining whether the compound binds to and activates orinhibits the binding by detecting the presence or absence of a signalgenerated from the interaction of the compound with the binding.
 22. Amethod for inducing an immunological response in a mammal whichcomprises inoculating the mammal with spsA, or a fragment or variantthereof, adequate to produce antibody to protect said animal from upperrespiratory tract (e.g. otitis media, bacterial tracheitis, acuteepiglottitis, thyroiditis), lower respiratory (e.g. empyema, lungabscess), cardiac (e.g. infective endocarditis), gastrointestinal (e.g.secretory diarrhoea, splenic abscess, retroperitoneal abscess), CNS(e.g. cerebral abscess), eye (e.g. blepharitis, conjunctivitis,keratitis, endophthalmitis, preseptal & orbital cellulitis,darcryocystitis), kidney and urinary tract (e.g. epididymitis,intrarenal and perinephric abscess, toxic shock syndrome), skin (e.g.impetigo, folliculitis, cutaneous abscesses, cellulitis, woundinfection, bacterial myositis) bone and joint (e.g. septic arthritis,osteomyelitis).
 23. A method of inducing immunological response in amammal which comprises, through gene therapy, delivering gene encodingspsA fragment or a variant thereof, for expressing spsA, or a fragmentor a variant thereof in vivo in order to induce an immunologicalresponse to produce antibody to protect said animal from disease.
 24. Animmunological composition comprising a DNA which codes for and expressesa spsA polynucleotide or protein coded therefrom which, when introducedinto a mammal, induces an immunological response in the mammal to agiven spsA polynucleotide or protein coded therefrom.